- The Gram stain technique was developed in 1884 by Danish physician Hans Christian Gram to classify bacteria based on differences in their cell walls. - Gram staining involves staining bacteria with crystal violet dye followed by treatment with iodine and decolorizing agent. Bacteria that retain the crystal violet stain are Gram-positive, while those that lose it and take up the counterstain are Gram-negative. - The key difference between Gram-positive and Gram-negative bacteria is the thickness and composition of their cell walls, with Gram-positive having a thicker peptidoglycan layer that retains the crystal violet iodine complex.

1. BACTERIAL STAINING
Dr. Muhammad Masood Ahmad

2. Hans Christian Joachim Gram
• The Gram stain was devised by
the Danish physician, Hans
Christian Joachim Gram, while
working in Berlin in 1883. He
later published this procedure in
1884. At the time, Dr. Gram was
studying lung tissue sections
from patients who had died of
pneumonia.

3. GRAM STAINING :
• DANISH BACTERIOLOGIST HANS
CHRISTIAN GRAM (1880)
• Based on this reaction, bacteria classified
into Gram positive and Gram negative
bacteria.
• The cell wall compostion differences
makes difference.

4. PRINCIPLE
•
•
•
The Gram Reaction is dependent on permeability of the bacterial cell wall and
cytoplasmic membrane, to the dye-iodine complex.
In Gram positive bacteria, the crystal violet dye –iodine complex combines to form
a larger molecule which precipitates within the cell. Also the alcohol/acetone
mixture which act as decolorizing agent, cause dehydration of the multi-layared
peptidoglycan of the cell wall. This causes decreasing of the space between the
molecules causing the cell wall to trap the crystal violet iodine complex within the
cell. Hence the Gram positive bacteria do not get decolorized and retain primary
dye appearing violet. Also, Gram positive bacteria have more acidic protoplasm
and hence bind to the basic dye more firmly.
In the case of Gram negative bacteria, the alcohol, being a lipid solvent, dissolves
the outer lipopolysaccharide membrane of the cell wall and also damage the
cytoplasmic membrane to which the peptidoglycan is attached. As a result, the
dye-iodine complex is not retained within the cell and permeates out of it during
the process of decolourisation. Hence when a counter stain is added, they take up
the colour of the stain and appear pink.

5. • A staining technique used to classify bacteria;
bacteria are stained with gentian violet and then
treated with Gram's solution; after being
decolorized with alcohol and treated with
safranine and washed in water, those that retain
the gentian violet are Gram-positive and those
that do not retain it are Gram-negative
Gram staining an Important
Technique

6. Gram positive Bacteria
• Gram positive bacteria
have a thick cell wall of
peptidoglycan and other
polymers. Peptidoglycan
consists of interleaving
filaments made up of
alternating
acetylmuramic acid and
acetylglucosamine
monomers.
In Gram positive
bacteria,between the cell
wall and cell membrane,
there is a "membrane
teichoic acid".

7. Gram Negative Bacteria
• Gram negative bacteria
have an outer membrane
of phospholipids and
bacterial
Lipopolysaccharides
outside of their thin
peptidoglycan layer. The
space between the outer
membrane and the
peptidoglycan layer is
called the periplasmic
space. The outer
membrane protects Gram
negative bacteria against
penicillin and lysozymes.

8. Constituents of the Cell wall
make difference

9. • Clean grease-free slide.
• Bacteria tobe stained.
• Inoculating loops- to transfer bacterial
suspension to slide.
• Bunsen burner – to sterilise inoculating
loops before and after smear preparation.
• Pencil marker – to mark (particularly
central portion of slide) where bacterial
smear is applied
Basic requirements for staining:

10. • Smear preparation:
• Putting of bacterial suspension (bacteria in
liquid) tobe stained on the central portion
of slide in a circular fashion, air-dried,
heat-fixed, the resultant preparation called
bacterial smear- appears dull white.
Basic initial steps before staining:

11. 1. Crystal violet – Primary stain
2. Gram’s iodine- mordant/fixative
3. Acetone (95%)- decoloriser
4. Safranine/dilute carbol fuchsin –
counterstain
REQUIREMENTS – STAINING REAGENTS:

12. Organizing the Staining Bottles

13. 1.Crystal violet - all bacteria take crystal violet- so
all appears violet.
2.Iodine – Crystal Violet-iodine(CV-I) complex is
formed.
3.Acetone- bacteria with high lipid content loose
CV-I complex(appear colouless) but bacteria with
less lipid content retains CV-I complex ( appear
violet).
4.Safranine/ dilute carbol fuchsin – only colouless
bacteria takes – appear pink.

14. PROCEDURE:
• Crystal violet – 1 min - wash.
• Iodine – 1 min – wash.
• Acetone add drop by drop and watch out colour
comes out – wash immediately.
• Safarnine/dilute carbol fuchsin – 1 min- wash.
• Allow to dry – examine under microscope.
Note: Results should be confirmed only with 100x.

15. • SMEAR FIXATION:
• 1) Heatfixation
• a) Pass air-dried smears through a flame two or three times. Do not
•
overheat.
b) Allow slide to cool before staining.
• 2) Methanol fixation
• a) Place air-dried smears in a coplin jar with methanol for one minute.
Alternatively, flood smear with methanol for 1 minute.
• b) Drain slides and allow to dry before staining.

16. Method of Picking material from
Agar plates
Wrong Right

17. Prefer to pick up colonies with loop
and smear on Clean glass slide

18. • i. Relative amounts of PMN’s, mononuclear
cells, and RBC's
• ii. Relative amounts of squamous epithelial cells,
bacteria
• consistent with normal micro flora, which may
indicate an improperly collected specimen
• iii. Location and arrangements of
microorganisms
• Note any special character sticks
Observe the Following Under Oil
Immersion lens

19. Colors makes the Difference in
Gram staining
• Bacteria that manage to
keep the original purple
dye have only got a cell
wall - they are called
Gram positive.
• Bacteria that lose the
original purple dye and
can therefore take up the
second red dye have got
both a cell wall and a cell
membrane - they are
called Gram negative.

20. RESULT:
Colour:
Purple colored bacteria – Gram positive
Pink colored bacteria – Gram negative
Shape:
Spherical – cocci
Rod – bacilli
Arrangement
Cocci in clusters – staphylococci
Cocci in chains - streptococci

21. • If no microorganisms are seen in a smear of a
clinical
• specimen, report “No microorganisms seen.”
• ii. If microorganisms are seen, report relative
numbers and
• Describe morphology.
• Observe predominant shapes of microorganisms
Report as follows

22. GRAM POSITIVE COCCI IN CLUSTERS

23. GRAM POSITIVE COCCI IN CHAINS

24. GRAM NEGATIVE BACILLI

25. GRAM POSITIVE BACILLI

26. GRAM NEGATIVE COCCI

27. Gram stain (Mixed)

28. • Gram positive cocci in clusters:
• 1. Staphylococci species.
• Gram negative cocci in chains:
• 1. Streptococci species.
• Gram negative cocci:
• 1. Neisseria species.
• Gram negative bacilli:
• 1. Escherichia coli
• 2. Klebsiella pneumoniae
EXAMPLES:

29. • Gram positive bacilli:
• 1. Clostridium species.
• 2. Corynebacterium species.
• 3. Bacillus anthracis.

30. Gram-negative Pseudomonas
aeruginosa bacteria (pink-rods).
30

31. (Bacillus anthracis),
31

32. Streptococcus pneumonia
32

33. Streptococcus salivarius

34. Corynebacterium diptheria seen in
Gram staining

35. Clostridium spp. seen in Gram Stain

36. Streptococcus pneumoniae in
Sputum

37. Gram stain of Neisseria
gonorrhoea,

38. Stains Several Fungi

39. Gram variable observations in
Gram staining
• The Gram staining procedure does not always
give clear-cut results. Some organisms are
Gram-variable and may appear either Gram-
negative or Gram-positive according to the
conditions. With these types of organisms,
Gram-positive and Gram-negative cells may be
present within the same preparation

40. Overcoming in Gram Variable
Observations
• it is necessary that it is stained at two or three
different ages (very young cultures should be
used). If an organism changes from positive to
negative or vice versa during its growth cycle,
this change should be recorded with a statement
as to the age of the culture when the change was
first observed. In case a Gram-variable reaction
is observed it is also good to check the purity of
the culture.

41. Why Gram Variability?
• Some Gram-positive bacteria appear Gram-
negative when they have reached a certain age,
varying from a few hours to a few days. On the
other hand, some Gram-negative bacteria may
become Gram-positive in older cultures. For this
reason it is strongly recommended to use very
young cultures for the staining procedure, after
growth has become just visible.

42. Trouble shooting in Gram
Staining method
The method and techniques used. Overheating
during heat fixation, over decolourization with alcohol,
and even too much washing with water between steps
may result in gram-positive bacteria losing the crystal
violet-iodine complex.
The age of the culture. Cultures more than 24 hours old
may lose their ability to retain the crystal violet-iodine
complex.
The organism itself. Some gram-positive bacteria are
more able to retain the crystal violet-iodine complex
than others.

43. •
•
By this method fairly good results may be obtained with very short staining
times, which are convenient when only one slide has to be stained.
Flood the slide with crystal or methyl violet stain and allow to act for about 5
seconds.
•
•
•
•
Tip off the stain and flood the tilted slide with iodine solution and allow to act
for about 5 seconds.
Tip off the iodine and flood the tilted slide
With acetone and allow this to act for only 2 seconds before washing it off
with water from the tap.
Flood the slide with basic fuchsin counter stain and allow it to act for about 5
seconds. Wash off with water, blot and dry.
QuickGRAM METHOD

44. Modification in Gram staining
methods
• Since the original procedure of Gram, many variations of the Gram
staining technique have been published. Some of them have improved
the method, others include some minor technical variants of no value.
• Bartholomew (1962) has pointed out that each variation in the Gram
staining procedure has a definite limit to its acceptability. Any final
result is the outcome of the interaction of all of the possible variables.
• All modified methods to be practised with caution should suit to the
laboratory, and quality control checks.

45. Various modifications of Gram staining
• 1. Kopeloff & Beerman’s Modification :
Primary stain is Methyl violet.
Decolourizer is Acetone/ Acetone-Alcohol mixture.
• 2. Jensen’s Modification :
Primary stain is Methyl violet.
Decolourizer is Absolute Alcohol.
•
Counter stain is Neutral Red.
3. Preston & Morrell’s Modification :
Primary stain is Crystal violet.
Decolourizer is Iodine-Acetone.
• 4. Weigert’s Modification:
Primary stain is Carbol Gentian violet.
Decolourizer is –Aniline-Xylol. Weigert stain is used to stain tissue sections.

46. Applications of Gram Staining
1.Rapid presumptive diagnosis of diseases such as Bacterial meningitis.
2.Selection of Empirical antibiotics based on Gram stain finding.
3.Selection of suitable culture media based on Gram stain finding.
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•
•
• 4.Screening of the quality of the clinical specimens such as sputum that
should contain many pus cells & few epithelial cells.
5.Counting of bacteria.
•
• 6.Appreciation of morphology & types of bacteria in clinical specimens.

47. THANK
YOU